Enzymic mechanism of starch breakdown in germinating rice seeds V. Sucrose phosphate synthetase in the scutellum

Some enzymic Properties of a partially purified preparationof sucrose phosphate synthetase (E.C.2.4.1.14) from germinatingrice seed scutella were studied. Examination of the reactionkinetics revealed that the rate of synthesis of sucrose phosphatefollows the Michaelis-Menten equation at an optimum PH of 7.5,having K_m of 25 mM for UDP-glucose, and of 4.9 mM for fructose6-phosphate. UDP inhibited the enzyme reaction competitively;K₁ of 3.3 mM. Fe⁺⁺ and Fe⁺⁺⁺ activated the enzyme reaction about2-fold; K_a, 0.3 mM and 2.0 mM, respectively. Co⁺⁺, Co(NH₃)₆⁺⁺⁺,Mg⁺⁺ and Mn⁺⁺ also activated the enzyme reaction. At high concentrationK⁺ activated the enzyme reaction with the maximum activationof 24% at 400 mM. The molecular weight and S_20,w value of theenzyme were determined as 4.5 ? 10⁵ and 10.4S, respectively. ¹Part IV of this series is Ref. (5). ²California Foundation for Biochemical Research Fellow (1973). (Received December 20, 1973; )     

Coupling of malate decarboxylation to CO2 fixation and the reduction of 3-phosphoglyceric acid in corn bundle sheath cells,2

1. In the presence of NADP⁺ and Mg⁺⁺, the bundle sheath strandsisolated from corn (Zea mays) leaves by cellulase treatmentsdecarboxylated malate in the light at an initial rate (200 µmoles/mgchl.hr), which was sufficient to account for photosyntheticCO₂ fixation in intact leaves. This rate gradually slowed downand then stopped. The final level of the malate decarboxylatedwas approximately equal to the amount of NADP⁺ added.
2. Rapidand continued decarboxylation of malate was observed whenNADP⁺,3-phosphoglyceric acid and ATP (and Mg⁺⁺) were addedtogether.The addition of ADP instead of ATP showed a similareffect.Light did not show any effect on the malate decarboxylationin the presence of ATP or ADP.
3. When malate was added to thebundle sheath strands in the presenceof exogenous NADP⁺ NADP⁺was rapidly reduced. The reductionstopped after 2 min when,73% of the added NADP⁺ was reduced.The further addition of3-phosphoglyceric acid and ATP broughtabout a decrease in theNADPH-level, which rose again to attaina new steady level.
4. The transfer of radioactivity from (1-¹⁴C-3-phosphoglycericacid to dihydroxyacetone phosphate in the bundle sheath strandsin the presence of ATP and NADP⁺ was greatly enhanced by theaddition of malate.
5. In the presence of ribose 5-phosphateand ATP, the rate of ¹⁴C-transferfrom (4-¹⁴C)-malate to theintermediates of the reductive pentosephosphate cycle was equalto that of ¹⁴CO₂ fixation in the light.

All these results support the current view that in the bundlesheath cells of C₄ plants belonging to the NADP-malic enzyme-group,the decarboxylation of malate is coupled to the fixation ofthe released CO₂ and the reduction of 3-phosphoglyceric acidformed as a result of CO₂ fixation. ¹ Part of this research was reported at the 40th Annual Meetingof the Botanical Society of Japan Osaka, December, 1975. ³ Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, 359 Otsuka, Hachioji-City, Tokyo 173, Japan. (Received April 30, 1977; )     

Electron transport dependent adenosine triphosphatase activity in castor bean endosperm mitochondria

The release of inorganic phosphate from ATP by mitochondriaisolated from endosperms of castor bean (Ricinus communis) wasstimulated by Mg⁺⁺, but not by Ca⁺⁺. EDTA, succinate, NADH₂or oligomycin depressed the reaction. The depression by succinatewas removed by KCN, antimycin A or anoxia. DNP alone did notaffect activity but did stimulate the Pi release in the presenceof succinate under aerobic conditions. Enhanced Pi release inthe presence of succinate and DNP was cancelled by KCN, antimycinA, oligomycin or anoxia. On the basis of these results, themechanism of ATPase action in castor bean endosperm mitochondriais discussed. (Received January 27, 1969; )     

The Organic Acid Metabolism of Bramley's Seedling Apple Peel1

1. The effect of various Krebs cycle acids on the respirationofdisks of apple peel at various stages of maturity was measuredin a Warburg respirometer.
2. Peel tissue from apples at thepre-climacteric and early post-climactericstages apparentlycontain sufficient of the Krebs cycle acidsused, with the exceptionof succinate, to maintain oxidativeprocesses at a maximum.
3. The addition of malate causes a large increase in the CO₂-outputof peel from post-climacteric and senescent fruit but not frompre-climacteric fruit, and a close correlation exists betweenthe climacteric and this decarboxylation of malate. The decarboxylationof malate does not affect the rate of O₂-uptake of peel tissue.The possible part played by the decarboxylation of malate inthe increased CO₂-output at the climacteric is discussed.
4. Addedpyruvate is decarboxylated by the tissue at all stagesof storagelife.
5. The decarboxylation of added malate is an aerobic fermentation,resulting in the quantitative production of acetaldehyde. Althoughthe presence of oxygen is necessary, the rate of O₂-uptake isnot affected by the reaction. Pyruvate decar boxylation doesnot require the presence of oxygen.
6. The O₂-uptake of peelfrom senescent apples can be stimulatedby addition of malate,succinate, and a-ketoglutarate. No evidencewas obtained, however,of oxidation of fumarate, citrate, orpyruvate. The additionof malate to senescent tissue restoresthe lower endogenousrate of O₂-uptake to that of early postclimacteric tissue.
7. Succinate and fumarate are toxic to peel tissue at concentrationabove 0.02M.

     

Effects of temperature and various metal ions on the solubility of tobacco RuDP carboxylase

Tobacco RuDP carboxylase is completely soluble in 0.07 M NaClor 0.01 M Na₂SO₂, but is almost completely insoluble in salt-freesolutions at 40°C; the solubility seeming to depend on ionicstrength. Lowering the temperature increased solubility of theprotein. The solubility in 0.01–0.04 M NaCl at 0°Cwas more than double that at 40°C. RuDP solubilized theprotein even in a salt-free medium. The protein became insolubleagain on the addition of various divalent cations. Effectivenessof the metal ions was Zn⁺⁺> Ni⁺⁺>Co⁺⁺>Mn⁺⁺>Mg⁺⁺>Ca⁺⁺.Although most of the metal ions inhibited (Mg⁺⁺ activated) enzymeactivity, no direct correlation was found between the degreeof solubility depression and the degree of enzyme inhibition. (Received October 4, 1971; )     

Kinetic Model of Mitochondrial Krebs Cycle: Unraveling the Mechanism of Salicylate Hepatotoxic Effects

This paper studies the effect of salicylate on the energy metabolism of mitochondria using in silico simulations. A kinetic model of the mitochondrial Krebs cycle is constructed using information on the individual enzymes. Model parameters for the rate equations are estimated using in vitro experimental data from the literature. Enzyme concentrations are determined from data on respiration in mitochondrial suspensions containing glutamate and malate. It is shown that inhibition in succinate dehydrogenase and α-ketoglutarate dehydrogenase by salicylate contributes substantially to the cumulative inhibition of the Krebs cycle by salicylates. Uncoupling of oxidative phosphorylation has little effect and coenzyme A consumption in salicylates transformation processes has an insignificant effect on the rate of substrate oxidation in the Krebs cycle. It is found that the salicylate-inhibited Krebs cycle flux can be increased by flux redirection through addition of external glutamate and malate, and depletion in external α-ketoglutarate and glycine concentrations.     

Modulation by Bicarbonate of Catalytic and Regulatory Properties of C4Phosphoenolpyruvate Carboxylase from Amaranthus hypochondriacus: Desensitization to Malate and Glucose 6-Phosphate and Sensitization to Mg2+

The catalytic and regulatory properties of phosphoenolpyruvate(PEP) carboxylase (PEPC) are modulated remarkably by the increasein the level of bicarbonate in the assay medium. The activityof PEPC increased by two-fold as the concentration of bicarbonatewas raised from 0.05 to 10 mM. During this state, there wasonly marginal effect on K_m for PEP, while the affinity of PEPCto Mg²⁺ increased by >2 fold. In contrast, the sensitivityof PEPC to malate decreased with increasing concentration ofHCO₃^–. Similarly, the stimulation by glucose 6-phosphate(G-6-P) at optimal concentration (10 mM) of HCO₃^– wasmuch less than that at suboptimal concentration (0.05 mM). K₁for malate increased by about 3 fold and K_a for G-6-P risedby fourfold as bicarbonate concentration was rised from 0.05to 10 mM. These results suggest that HCO₃^– desensitizesPEPC to both malate and G-6-P. Further, these changes were manifestedin both dark- as well as light-forms of the enzyme. Similarresults were obtained with PEPC in leaf extracts or in purifiedform. We therefore propose that bicarbonate-induced changesare independent of phospho-rylation and possibly through a significantchange in the conformation of the enzyme. This is the firstdetailed report indicating marked modulation of regulatory andcatalytic properties of PEPC by bicarbonate, one of its substrate. (Received April 14, 1998; Accepted September 22, 1998)     

ENZYMIC FORMATION OF GLUTAMINE IN RICE-PLANT SEEDLINGS

1. Purified preparation from rice-plant seedling catalyses a stoichiometricreaction between ATP, glutamate, and NH₂OH in the presence ofMg⁺⁺ to form glutamyl hydroxamate, ADP and inorganic phosphate.
2. The method of purification and some of the properties of theenzyme are described. Co⁺⁺ can be substituted for Mg⁺⁺. Mn⁺⁺,NaF, and PCMB inhibit the enzyme strongly.
3. Inorganic orthophosphateis liberated from ATP by the additionof or cysteine in the presence of glutamate, Mg⁺⁺ andthis preparation. Glutamine was detectedin the reaction productsby paperchromatography.
4. The same preparation catalyses a reactionbetween gluta mineand NH₂OH in the presence of Mg⁺⁺ or Mn⁺⁺,ADP and inorganicphosphate, to form glutamyl hydroxamate.

¹ Present address: The Department of Chemistry, Faculty of Science,Kanazawa University, Kanazawa. (Received October 31, 1960; )     

Regulation of Steady-State Photosynthesis in Isolated Intact Chloroplasts under Constant Light: Responses of Carbon Fluxes, Metabolite Pools and Enzyme-Activation States to Changes of Electron Pressure

The reactions of isolated intact spinach chloroplasts at saturatinglight and CO₂ to changes in steady-state electron flow werefollowed at the various stages of photosynthesis. Alterationsin the rate of electron flow were induced by the addition ofoxaloacetate (OAA), nitrite or methyl viologen (MV). Two typesof effect can be distinguished: (1) When a small fraction ofthe electrons produced are accepted by OAA or nitrite (up to20% of the electrons produced in the light), the activationstate of the NADP⁺-dependent malate dehydrogenase (NADP-MDH)was strongly decreased, whereas qP and the rate of O₂-productionwere increased. qN, the stromal metabolite pools and the [¹⁴C]-CO₂-fixationrate were only marginally influenced. (2) Higher amounts ofnitrite or MV decreased O₂ production and strongly inhibited[¹⁴C]CO₂ fixation. This treatment further increased the ATP/ADPratio, but had little effect on the NADPH + H⁺/NADP⁺ ratio.The stromal concentrations of 3PGA, DHAP and FBP, and the ratesof 3PGA and DHAP export were drastically changed. In particular,the DHAP/3PGA ratio increased, and the rate of 3PGA export wasdecreased by minor changes in the rate of electron flow. Additionof high amounts of nitrite or MV, but not of OAA decreased theactivation states of NADP-MDH and fructose 1,6-bisphosphatase(FBPase), while the activation states of NADP⁺-dependent glyceraldehyde3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK)remained unchanged under all conditions. (Received February 10, 1997; Accepted September 2, 1997)     

Ribulose-1, 5-diphosphate carboxylase of Chromatium strain D

RuDP carboxylase isolated from autotrophically grown cells ofphotosynthetic sulfur bacterium, Chromatium strain D, was partiallypurified by (NH₄)₂SO₄ precipitation and Sephadex G-200 gel filtration.The molecular size of the bacterial RuDP carboxylase was foundto be large, analogous to that of the plant enzyme, in agreementwith results of previous workers. Sucrose density gradient centrifugationshowed the S_rel to be approximately 18; the omission of Mg⁺⁺caused no dissociation of the enzyme molecule in its subunits.Chromatium RuDP carboxylase showed similarities to the plantenzyme in some of its kinetic properties; (a) a shift of pHoptimum to the neutral side from the alkaline side on the additionof Mg⁺⁺, (b) deviation of the substrate concentration (NaHCO₃)-activityrelationship from the MICHAELIS formula and (c) a marked stimulativeeffect of Mg⁺⁺. A unique sigmoidal saturation curve of the enzymeto RuDP, which had been detected in Rhodospirillum rubrum andRhodopseudomonas spheroides RuDP carboxylase in the absenceof Mg⁺⁺, was not found. Another characteristic feature of ChromatiumRuDP carboxylase is its partial immunological response to therabbit anti-spinach RuDP carboxylase serum as detected by theinhibition of the carboxylation reaction due to the antibody-antigenreaction. ¹Part X, Structure and Function of Chloroplast Proteins. Supportedin part by research grants from the Ministry of Education ofJapan (No. 8719) and USPHS (AM-10792-03) (Received July 4, 1969; )     

Studies on the phosphomannose isomerase of Amorphophallus konjac C. Koch II. Effect of divalent metal ions on the EDTA-treated enzyme

Konjak phosphomannose isomerase was inactivated in a time-dependentprocess by metal binding agents, and the inactivated enzymewas instantaneously reactivated by adding such metal ions asZn⁺⁺, Co⁺⁺, Fe⁺⁺, Mn⁺⁺ and Cu⁺⁺. However, neither Ca⁺⁺ nor Mg⁺⁺were effective for reactivation. Zn⁺⁺, at a low concentration,brought about complete reactivation of the enzyme at pH 6–7. The EDTA-treated enzyme was more susceptible to heat denaturationwhen compared with the native enzyme, but the addition of variousmetal ions caused the recovery of the thermal stability of theEDTA-treated enzyme. The magnitude of the recovery dependedon the metal ion species and the concentrations. The most effectivemetal ion was Co⁺⁺, which caused the recovery of thermal stabilityto a level higher than that of the native enzyme. Phosphomannoseisomerase was inhibited by pchloromercuribenzoate and HgCl₂;the inhibition by p-chloromercuribenzoate being more pronouncedas incubation progressed. In contrast, the EDTA-treated enzymewas more readily inhibited by the mercurial ion than was thenative enzyme. Zn⁺⁺, when added to the EDTA-treated enzyme,markedly restored its resistance to the mercurial-induced inhibition.The metal-substituted enzyme was also inhibited by EDTA in atime-dependent process. ¹ This paper constitutes part 4 of studies on konjak mannanbiosynthesis. (Received March 3, 1975; )     

Metal Interrelationships in Plant Nutrition: 2. THE RELATION OF METAL TOXICITY, MOLYBDENUM AND NITROGEN SOURCE TO CHLOROPHYLL AND MAGNESIUM CONTENT OF BEET IN SAND CULTURE

Sugar beet and spinach beet were grown in sand culture withdifferent sources of nitrogen or iron and different levels ofmolybdenum and heavy metals. Chlorophyll and some magnesiumfractions were determined. Extra molybdenum accentuated chlorosis caused by excess of Mn⁺⁺,Cr⁺⁺⁺, Zn⁺⁺, and Cu⁺⁺ and decreased it in the presence of CrO₄^––with nitrate. In the presence of these ions urea reduced chlorophyll in youngand increased it in old leaves of sugar beet. Effects of ureaand molybdenum were additive and independent. Ammonium sulphatecaused increased chlorosis of spinach beet with Mn⁺⁺ and Zn⁺⁺. The existence of an acetone-soluble magnesium fraction otherthan chlorophyll was shown. Excess Cu⁺⁺ and Zn⁺⁺ decreased totalmagnesium; Mn⁺⁺ did not. Mn⁺⁺ and Zn⁺⁺ reduced chlorophyll,increased the non-chlorophyll acetone-soluble magnesium, butreduced other fractions. Ferrous iron ipcreased total magnesiumcontent and also increased the acetone-soluble fractions inthe presence of Mn⁺⁺ and Zn⁺⁺. Extra molybdenum decreased theacetone-soluble magnesium fractions but did not affect the totalmagnesium content. Significant interactions between Mo, N, Fe, and heavy metalswere also disclosed.     

The Effect of Subjecting Peas to Air Enriched with Carbon Dioxide: II. RESPIRATION AND THE METABOLISM OF THE MAJOR ACIDS

Whole peas at about 75 per cent of their maximum fresh weightwere subjected to 5–30 per cent CO₂ in air for periodsof from 1–6 d, then returned to air for a further 1 d.Samples were withdrawn at intervals and organic acids, ^TCO₂and ethanol estimated as well as the rate of respiration. Slicesof cotyledons suspended in water were also subjected to highconcentrations of CO₂ in air for 3 h. The rate of respiration was inhibited progressively by increasein CO₂ content of the tissue. The high internal CO₂ contentof the intact pea causes an inhibition of its rate of respirationby about 25 per cent. Alcohol production commenced at between10 and 15 per cent CO₂ in the ambient gas and slowly increasedin rate up to 37 per cent. The CO₂-air mixtures reduced the content of malate, pyruvateand -oxoglutarate, increased that of succinate and left citrateunaffected. On return to air malate rose rapidly and succinatefell slowly to their original concentrations. During the sameperiod the concentration of PEP fell sharply and after about1 h rose again, whereas oxalacetate showed a reverse response.It is argued that the rapid re-synthesis of malate was by carboxylationof PEP to oxalacetate and that this reaction was stimulatedby a change in pH rather than by the direct effect of the changein concentration of CO₂. In one experiment ¹⁴CO₂ was supplied for 2 h before return toair and the movement of ¹⁴C followed for 6 h. The results supportthe method of re-synthesis of malate proposed.     

Effect of Root Zone Temperature on the Mineral Composition of Xylem Sap and Plasma Membrane K+-Mg++-ATPase Activity of Grafted-Cucumber and -Figleaf Gourd Root Systems

Cucumber (Cucumis sativus L.) seedlings were grafted onto cucumber-(CG) or figleaf gourd- (FG, Cucurbita ficifolia Bouché)seedlings in order to determine the effect of solution temperature(12, 22, and 32°C) on the mineral composition of xylem sapand the plasma membrane K⁺-Mg⁺⁺-ATPase activities of the roots.Low solution temperature (12°C) lowered the concentrationof NO^–₃ and H₂PO^–₄ in xylem sap of CG plants butnot of FG plants. Concentrations of K⁺, Ca⁺⁺ and Mg⁺⁺ in xylemsap were less affected than anions by solution temperature.The plasma membrane of FG plants grown in 12°C solutiontemperature showed the highest K⁺- Mg⁺⁺-ATPase activity at allATP concentrations up to 3 mM and at low reaction temperatureup to 12°C, indicating resistance of figleaf gourd to lowroot temperature. (Received December 27, 1994; Accepted March 10, 1995)     

Progress No. 9 Cultivar of Pea Has Alternative Respiratory Capacity and Normal Glycine Metabolism

Mitochondria from the dwarf pea cultivar Progress No. 9, havebeen reported to lack alternative respiration. However, therates of respiration with succinate and malate by mitochondriaisolated from Progress No. 9, although approximately 30% lowerthan those from Alaska, had the same percentage distributionof respiratory capacity between the cytochrome pathway and thealternative pathway. Immunoblots showed both cultivars containpolypeptides identified as components of the alternative oxidase.Both cultivars formed identical products during photosynthetic¹⁴CO₂ fixation, and the percentage distribution of ¹⁴C intoglycine and serine were similar. In spite of large amounts ofglycine decarboxylase in the isolated leaf mitochondria, ratesof O₂ uptake with glycine were similar to rates with malateor succinate. It appears that glycine oxidation was coupledto the alternative oxidase to the same extent as malate andsuccinate oxidation, and the alternative oxidase was not specificallyutilized for glycine oxidation. (Received May 21, 1990; Accepted December 19, 1990)     

The Effects of Gibberellin on the Metabolism of Ethanol-soluble Constituents in the Cotyledons of Hazel Seeds (Corylus avellana, L.)

Gibberellin pretreatment of hazel seeds induced significantincreases in the incorporation of carbon-14 from [2-¹⁴C] acetateinto both glutamate and sucrose, while the amounts of radioactivityincorporated into citrate, malate and succinate were significantlyhigher after pretreatment with sterile water. A ratio basedon the relative amounts of carbon-14 incorporated into glutamateand into the TCA cycle acids indicates that gibberellin treatmentcauses a diversion of metabolites from the essentially respiratoryTCA cycle into synthetic sequences such as glutamate formation.Gibberellin treatment also appears to stimulate the activityof a reversed glycolytic sequence, resulting in the productionof sucrose.     

RESPIRATORY METABOLISM IN THE PHLOEM, XYLEM AND CAMBIUM OF CARROT ROOT

Comparisons of respiratory metabolism among the phloem, cambiumand xylem of mature carrot root were carried out and the followingresults were obtained. 1) The activity of O₂ uptake on a dry weight basis in the cambiumwas higher than that in the xylem. The phloem showed the lowestactivity. 2) RQ was close to unity in all the three tissues.3) The inhibition rate of O₂ uptake by malonate was considerablyhigh in the three tissues. The highest inhibition was observedin the cambium. 4) In the phloem and xylem the malonic inhibitionrate of ¹⁴CO₂ release from G-U-¹⁴C was not so high as comparedwith that of O₂ uptake. In the cambium, however, those of O₂uptake and ¹⁴CO₂ output were comparable. 5) Malonate treatmentdid not cause any significant change in RQ. 6) The C₆/C₁ ratiowas highest in the cambium and lowest in the phloem. From these it is concluded that the participation of the TCAcycle in respiration is great in all the three tissues of carrotroot, and among the three the cambium has the highest activityof the cycle. As for the PP pathway, the xylem and phloem havea higher activity than the cambium. (Received April 11, 1967; )     

Movement of Ions and Electrogenesis in Higher Plant Cells

During the past 10 years considerable information has accumulatedon the electrochemical relationships of higher plant cells duringtransport of mineral ions. Using the Nernst equation as a criterion,none of eight ions (K⁺, Na⁺, Ca⁺⁺, Mg⁺⁺, NO₃^–, Cl^–,H₂PO₄^–, and SO₄^–) is in a passive equilibrium. Na⁺,Ca⁺⁺, and Mg⁺⁺ are subject to an exclusion mechanism, and allof the anions appear to be pumped inwardly. K⁺ apparently approachesan electrochemical balance under certain conditions but probablyis actively accumulated. Compartmental analyses giving estimatesof amounts in the cytoplasm and vacuole and of unidirectionalfluxes permit application of the Ussing flux-ratio equation.The criterion in oat coleoptile cells suggests that at the plasmalemmaNa⁺ is pumped out while K⁺ and Cl^– are pumped in. K⁺ andCl^– appear to be coupled in active transport across thetonoplast into the vacuole. Good evidence has been found thatthe cell's electropotential arises from an electrogenic pump:CN^– (cyanide) and DNP (dinitrophenol) reversibly blockthe potential and ionic transport; cell potentials are higherthan can be accounted for by diffusion; the responses of respirationand potential to the concentration of CN^– are nearly parallel;and CN^– inhibited tissue approaches a fit to the Goldmanconstant field equation. Future objectives should be identificationof the ion, or ions, subject to the electrogenic pump and discoveryof the immediate energy source.     

The Effect of Carnitine on Palmitate Oxidation by Pea Cotyledon Mitochondria

The conditions for maximum O₂ uptake by pea cotyledon mitochondriaoxidizing palmitate were established. It was found that CoASH,Mg²⁺, ATP, malate, and carnitine were necessary additions tothe incubation medium. It is suggested that carnitine combineswith palmitoylCoA to produce palmitoylcarnitine with the releaseof CoASH. The palmitoylcarnitine thus formed may penetrate themembranes of the mitochondria with greater ease than palmitoylCoA.     

Sodium gradient-dependent transport of magnesium in rat ventricular myocytes

Cytoplasmic concentration of Mg²⁺([Mg²⁺]_i) was measured with a fluorescentindicator furaptra in ventricular myocytes enzymatically dissociatedfrom rat hearts (25°C). To study Mg²⁺ transport acrossthe cell membrane, cells were treated with ionomycin inCa²⁺-free (0.1 mM EGTA) and high-Mg²⁺ (10 mM)conditions to facilitate passive Mg²⁺ influx. Rate of riseof [Mg²⁺]_i due to the net Mg²⁺influx was significantly smaller in the presence of 130 mMextracellular Na⁺ than in its absence. We also tested theextracellular Na⁺ dependence of the net Mg²⁺efflux from cells loaded with Mg²⁺. After[Mg²⁺]_i was raised by ionomycin and highMg²⁺ to the level 0.5-0.6 mM above the basal value(~0.7 mM), washout of ionomycin and lowering extracellular[Mg²⁺] to 1.2 mM caused rapid decline of[Mg²⁺]_i in the presence of 140 mMNa⁺. This net efflux of Mg²⁺ was completelyinhibited by withdrawal of extracellular Na⁺ and waslargely attenuated by imipramine, a known inhibitor of Na⁺/Mg²⁺ exchange, with 50% inhibition at 79 µM. The relation between the rate of net Mg²⁺ efflux andextracellular Na⁺ concentration([Na⁺]_o) had a Hill coefficient of 2 and[Na⁺]_o at half-maximal rate of 82 mM. Theseresults demonstrate the presence of Na⁺ gradient-dependentMg²⁺ transport, which is consistent withNa⁺/Mg²⁺ exchange, in cardiac myocytes.